1. Field of the Invention
This invention relates to new hybridomas and monoclonal antibodies therefrom which recognize distinct antigens on subpopulations of chicken T-lymphocytes.
2. Abbreviations
Abbreviations or definitions used in the disclosure are as follows: BSA--bovine serum albumin; C--rabbit complement; ConA--concanavalin A; CTL--cytotoxic T lymphocytes; CTLA--chicken T lymphocyte antigen; DTT--dithiothreitol; EDTA--ethylene diamine tetraacetic acid; ELISA--lymphocyte binding enzyme linked immunosorbent assay; E:T--effector to target; FCM--flow cytometric; FCS--fetal calf serum; HBSS--Hanks balanced salt solution; IEL--intraepithelial lymphocytes; LGL--large granular lymphocytes; MAbs--monoclonal antibodies; MDV--Marek's disease virus; MHC--major histocompatibility complex; NK--natural killer; PBL--peripheral blood lymphocytes; PBS--phosphate buffered saline; PEG--polyethylene glycol; REV--reticuloendotheliosis virus; SD--standard deviation; SR--% spontaneous release; TR--total release.
3. Summary of Prior Art
The immune system of chickens has received much attention as a model for studying lymphocyte differentiation and human immunodeficiency states [Blaese et al., In Avian Immunology, A. A. Benedict (ed.), Plenum Publishing Co., New York, p. 155 (1977); Palladino et al., J. Immunol. 116:1673 (1976)]. However, although modern hybridoma technology has made it possible to separate mouse and human lymphocytes into functional subsets [Cantor and Boyse, J. Exp. Med. 141:1376-1389 (1975); Engleman et al., Proc. Natl. Acad. Sci. USA 78:1791-1795 (1981)], monoclonal antibodies defining subsets of chicken lymphocytes have not been generally available. Until recently, surface antigens distinguishing chicken thymus- and bursa-derived lymphocytes were detected using heteroantisera [Wick et al., Clin. Exp. Immunol. 15:237-249 (1973)]. The limitations of both allo- and xenoantisera can now be resolved by the generation of MAbs with the somatic cell hybridization techniques introduced by Kohler and Milstein [Nature 256:495 (1975)].
In mammals, a series of MAbs detecting surface antigens has enhanced our knowledge of functionally distinct subpopulations of lymphocytes [Kung et al., Science 206:347 (1979); Reinherz et al., J. Immunol. 123:2894 (1979); Reinherz et al., Proc. Natl. Acad. Sci. USA 77:1588 (1980); Ledbetter et al., J. Exp. Med. 153:319 (1981); Dialynas et al., J. Immunol. 131:2445 (1983)]. In chickens, subpopulations of chicken T lymphocytes with distinct helper [Grebenau et al., Eur. J. Immunol. 9:477 (1979); Sarvas et al., Scand. J. Immunol. 3:455 (1974); Chi et al., Eur. J. Immunol. 10:23 (1980)], suppressor [Chi et al., supra; Grebenau et al., supra; Lerman et al., Cell Immunol. 51:109 (1980)], or cytotoxic [Palladino et al., Dev. Compar. Immunol. 4:309 (1980); Chi et al., Cell Immunol. 64:246 (1981); Maccubin and Schierman, J. Immunol. 136:12 (1986)] activities have been described. Although cell surface antigens detecting subpopulations of chicken T lymphocytes have been detected by xenoantisera or alloantisera, only in a few cases have the relevant antigens been biochemically or functionally characterized [Peault et al. Eur J. Immunol. 12:1047 (1982); Pink and Rijnbeek, Hybridoma 2:287 (1983); Houssaint et al., Eur. J. Immunol. 15:385 (1985)]. Some antigenic markers associated with subpopulations of avian T lymphocytes have recently been described [Chan et al., J. Immunol. 140:2133 (1988)], and analogies were drawn between the human and chicken lymphocyte antigens.
Four systems of T cell differentiation alloantigens have been reported in the chickens. They are Th-1 [Gilmour et al., Immunogenetics 3:549-563 (1976)], Ly-4[Fredrickson et al., Immunogenetics 5:535-552 (1977)], CA1 and TA [Galton and Ivanyi, Eur. J. Immunol. 7:241-246, 457-459 (1977)]. These antigens were detected using polyclonal antisera absorbed against bursa lymphocytes [Gilmour et al., supra]. Only a few monoclonal antibodies recognizing antigens on chicken T cells are available [Chen et al., Eur. J. Immunol. 14:385 (1984); Hala et al., Immunobiology 168:2 (1985); Houssaint et al., supra; Lillehoj, In Avian Immunology, Weber and Ewert (eds.), pp. 87-97, Alan R. Liss, Inc., New York (1987)], and most of these antibodies detect antigens expressed primarily on thymocytes. Two monoclonal antibodies that identify cell-surface antigens expressed on functionally distinct T-cell subpopulations in birds have been reported [Chan et al., supra]. Monoclonal antibodies detecting surface alloantigens of chicken lymphocytes will overcome the problems associated with the use of polyspecific antisera, such as the presence of antiviral antibodies and autoantibodies, difficulty in reproducing antisera and obtaining large amounts, and need to absorb sera to obtain required specificity.
The monoclonal antibodies described in this invention will allow the poultry industry and scientists working with poultry to isolate and investigate important subpopulations of lymphocytes for assessing immune status of the flock during infections or following vaccination. Since these monoclonal antibodies identify defined antigens or lymphocytes, the investigation of the role of these antigens in economically important diseases of poultry will now be possible.